The rat pheochromocytoma cell line PC12 has been extensivelyused as a model system for studying mechanisms involved in neuronalcell differentiation (1)
. In response to NGF,2
PC12 cellscease division and differentiate into sympathetic neuron-likecells with extensive neurites (2)
. PC12 cells also expressreceptors for growth factors such as fibroblast growth factorand EGF. Despite similarities in the tyrosine phosphorylationof different substrates, NGF and EGF induce markedly differenteffects on PC12 cells, with EGF inducing cell proliferationrather than differentiation (3)
. Binding of NGF and EGF totheir respective receptors induces receptor autophosphorylationand activation of overlapping second messenger pathways (4, 5). The basis for the distinct cellular outcomes induced byNGF and EGF is not fully understood (6)
. One major differencebetween NGF and EGF is the more prolonged activation in responseto NGF of different kinases such as MAPK (7, 8)
and phosphatidylinositol3'-kinase (9)
. In addition, certain proteins such as SNT havebeen shown to be phosphorylated specifically in response toNGF but not to EGF and have therefore been implicated in theability of NGF to induce neuronal differentiation (10)
. AlthoughNGF activates PKC in PC12 cells (11, 12)
, and PKC has beenimplicated in PC12 cell differentiation (13, 14)
, it is currentlynot known whether NGF and EGF differ in their effects on PKCactivity.

PKC is a family of phospholipid-dependent serine-threonine kinasesinvolved in the signal transduction of many physiological stimuli(15)
. PKC is activated by DAG, a second messenger that is generateddirectly when phospholipase C is activated by extracellularstimuli including hormones and growth factors or indirectlyvia phosphatidic acid formation by phospholipase D (16, 17)
.PKC is also the receptor for the potent tumor-promoting phorbolesters, which can substitute for DAG in PKC activation (18)
.PKC plays a prominent role in the regulation of cell proliferationand differentiation (19)
. Eleven isoforms of PKC have beenisolated thus far, showing characteristic patterns of distributionin different tissues (20)
. The unique expression of specificPKC isoforms in different tissues and cells suggests that differentisoforms fulfill distinct functions, and numerous studies arebeginning to dissect these isoform-specific roles in differentsystems. The PKC isoforms are divided into three main groupsbased on sequence homology and similarity in biochemical properties.The conventional PKCs (, ß1, ß2, and ) are Ca2+-dependentand are regulated by DAG and phorbol esters. The novel PKCs(, , , and ) are Ca2+-independent but show similar responsesto DAG and phorbol esters. In contrast, the atypical PKCs (and /) are insensitive to Ca2+, DAG, and phorbol esters. Finally,PKC-µ shows multiple differences relative to the otherPKCs (21)
.

In this study, we compared the effects of NGF and EGF on theactivation of specific PKC isoforms to determine whether differentialactivation of specific PKC isoforms might contribute to thedifferences in the effects of these two growth factors on neuriteoutgrowth. Our data indicate that PKC-, -, and - are differentiallytranslocated by NGF and EGF treatments. We further show thatEGF induces neurite outgrowth, as contrasted with proliferation,in cells overexpressing PKC-.

Differential Translocation of PKC Isoforms by NGF and EGF in PC12 Cells.
We first analyzed the expression and cellular distribution ofPKC isoforms in the PC12 cells used in our study and their responsesto NGF and EGF treatment. Fig. 1
shows that these PC12 cellsexpress PKC-, -ß, -, -, -, -µ, and - as determinedby Western blot analysis using specific PKC isoform antibodies.Treatment of the cells with NGF for 15 min induced a partialtranslocation of PKC- and - to the membrane, the translocationof PKC- to the membrane and the cytoskeleton, and the translocationof PKC- to the cytosol and the nucleus (data not shown). Theeffect of EGF on PKC- and - was similar to that of NGF. On theother hand, EGF induced only a partial translocation of PKC-to the membrane and did not affect the translocation of PKC-.EGF also induced the translocation of PKC- to the membrane,whereas NGF exerted a smaller effect.

Fig. 1. Expression and translocation of PKC isoforms in NGF- and EGF-treated PC12 cells. Aliquots of soluble (S), membrane (M), and cytoskeletal (C) fractions of PC12 cells untreated or treated with NGF or EGF for 15 min were subjected to SDS-PAGE and immunoblotted as described in "Materials and Methods." Blots were probed with antibodies specific to the appropriate PKC isoforms, as indicated in the figure. The results shown are from one of four representative experiments.

Overexpression of PKC Isoforms.
Because major differences were observed in the translocationof PKC- and - by NGF and EGF, we wanted to further examine thepossible role of these isoforms in mediating the biologicaleffects of NGF and EGF. Our approach was to use PC12 cells overexpressingPKC- or -; for comparison, we also included PC12 cells overexpressingPKC-.

Using the MTH vector, we transfected PC12 cells with PKC-, -,and -. To examine the level of protein expression, we analyzedthe pooled cultures and three different overexpressing clonesfor each of the isoforms by Western blotting and compared themwith the vector controls. Fig. 2
illustrates a representativeWestern blot of PC12 cells overexpressing the different PKCisoforms and the vector control. The levels of the overexpressedPKC-, -, and - in the transfected cells were 710-foldhigher than the corresponding endogenous PKCs, as determinedusing isoform-specific antibodies (Fig. 2)
. To establish thatthe overexpressed PKC isoforms were functionally active, wemeasured specific [3H]PDBu binding on partially purified celllysates. All PKC clones exhibited increased [3H]PDBu bindingas compared to the control transfected cells (Table 1)
. Moreover,binding was further enhanced in cells treated for 24 h with20 µM ZnCl2, an inducer of the metallothionein expressionvector (data not shown).

Fig. 2. Overexpression of PKC isoforms in PC12 cells. Stable transfectants of PC12 cells overexpressing the different PKC isoforms or the control vector were harvested and subjected to SDS-PAGE and Western blot analysis. The membranes were probed with specific anti-PKC-, -, or - antibodies. The immunoreactive bands were visualized as described in "Materials and Methods." The results shown are of one representative experiment of pooled clones; similar results were obtained in each of five additional experiments that were performed with three different clones.

Morphology and Proliferation of PC12 Cells Overexpressing Different PKC Isoforms in Response to EGF and NGF.
The morphology of the PC12 cells overexpressing PKC- or - wassimilar to the empty vector control cells and the parental PC12line (data not shown). Likewise, the proliferation of thesecells was not significantly different (Fig. 3)
. In contrast,cells overexpressing PKC- showed slower cell growth comparedto the empty vector control cells (Fig. 3)
, and some of thesecells extended small processes (Fig. 4D)
. NGF induced neuriteoutgrowth in all of the PKC-overexpressing cell lines. However,the degree of neurite outgrowth, as reflected by their lengthand by the number of cells extending neurites, was significantlyhigher in cells overexpressing PKC- compared to the empty vectorcontrol cells or to the other PKC overexpresser cell lines (Fig.4
; Table 2
). Treatment of empty vector control cells or cellsoverexpressing PKC- or - with EGF did not induce significantchanges in the morphology of the cells. As determined by BrdUrdincorporation, treatment of the cells with EGF for 4 days increasedcell proliferation by 56 ± 7.1% (P < 0.005) in vectorcontrol cells, by 46 ± 6.2% (P < 0.002) in cells overexpressingPKC-, and by 97 ± 9.2% (P < 0.01) in cells overexpressingPKC-, whereas it exerted a small inhibitory effect (35 ±2.4%; P < 0.001) on the proliferation of cells overexpressingPKC- (Fig. 3)
. Similar results were obtained in parallel experimentsin which cells were counted (data not shown). Likewise, similarresults were obtained with an incubation time of 6 days (datanot shown). Unlike the results obtained with the other PKC isoform-overexpressingcells, the cells overexpressing PKC- showed a striking differencein their morphological response to EGF. In these cells, EGFinduced marked neurite outgrowth (Fig. 4F)
. The kinetics ofneurite outgrowth in EGF-treated cells was similar to that ofthe NGF-treated cells. However, neurites of the EGF-treatedcells were less arborized than those seen in NGF-treated cells,and fewer cells with multiple neurites (more than three) wereobserved.

Fig. 3. Proliferation of PC12 cells overexpressing PKC-, -, and -. Cells were plated in 96-well plates, and cell proliferation was determined after 4 days in culture. BrdUrd was added to the cells for the last 6 h, and the assay was performed as described in "Materials and Methods." Background readings were subtracted, and the results are expressed in optical density (OD) units. The results represent the mean ± SE of three separate experiments.

Fig. 4. Morphology of NGF- and EGF-treated vector control PC12 cells and cells overexpressing PKC-. Stable transfectants of PC12 cells overexpressing PKC- or the control vector were cultured for 4 days on collagen-coated dishes in the absence and presence of NGF (50 ng/ml) or EGF (10 ng/ml). Cells were counted and scored for the presence of neurites. The results represent one representative experiment of five similar experiments.

Table 2 Percentage of cells with neurites in cells overexpressing different PKC isoforms after treatment with NGF and EGF

The neurite outgrowth induced by EGF was inhibited in cellspretreated with the PKC inhibitor GF 109203X (1 µM), consistentwith the effect of EGF being associated with PKC activation(data not shown).

EGF Induces Sustained Phosphorylation of MAPK in PC12 Cells Overexpressing PKC-.
One of the marked biochemical differences between the effectsof NGF and EGF on PC12 cells is the kinetics of phosphorylationand activation of MAPK. NGF induces a sustained activation ofMAPK, whereas EGF induces only a transient activation of thiskinase (7, 22)
. We therefore examined the pattern as a functionof the time of MAPK phosphorylation by NGF and EGF in cellsoverexpressing PKC-.

NGF induced maximal phosphorylation of both ERK1 and ERK2 after5 min, and this phosphorylation persisted for at least 1 h.In contrast, as described previously by others (7)
, EGF induceda transient phosphorylation of ERK1 and ERK2 that declined after15 min. Similar patterns of response to NGF and EGF were observedin cells overexpressing PKC- and - (data not shown). In contrast,cells overexpressing PKC- showed an enhanced activation of bothERK1 and ERK2 in response to NGF as compared to the controlvector cells. Overexpression of PKC- dramatically increasedthe phosphorylation of ERK1 and ERK2 by EGF and caused the responsein these cells to persist for up to 1 h (Fig. 5)
.

Fig. 5. EGF- and NGF-induced MAPK phosphorylation in PC12 cells overexpressing PKC-. Cells were stimulated with NGF (A) or EGF (B) for the time periods indicated. ERK1 and ERK2 phosphorylation was detected by Western blot analysis using a phospho-MAPK antibody. The results represent one representative experiment of five similar experiments. Similar results were obtained with three additional clones of PKC--overexpressing cells.

The MAPK Kinase Inhibitor PD98059 Inhibits EGF-induced Neurite Outgrowth in Cells Overexpressing PKC-.
To examine the role of MAPK activation in the induction of neuriteoutgrowth by EGF in the PKC- overexpressers, we pretreated thecells with the MAPK kinase inhibitor PD98059 (23)
for 1 h,followed by treatment of the cells with EGF. As presented inFig. 6
, treatment of the cells with PD98059 caused a 75 ±8.3% decrease in the percentage of neurite-bearing cells, thussuggesting that the prolonged activation of MAPK by EGF in thePKC- overexpressers plays a role in the induction of neuriteoutgrowth by EGF.

Fig. 6. PD98059 inhibits EGF-induced neurite outgrowth in cells overexpressing PKC-. PC12 cells overexpressing PKC- were treated with PD98059 (10 µM) for 1 h, followed by treatment with EGF (10 ng/ml) for 3 days. Cells were counted and scored for the presence of neurites. Values represent the mean values ± SE of quadruplicate determinations from each of three separate experiments. The results were reproduced with three additional clones of PKC-.

Cells Overexpressing a PKC- Dominant Negative Mutant.
Our results suggest an important role for PKC- in the processof neurite outgrowth induced by both NGF and EGF. To furtherexamine the role of this isoform, we transfected cells witha PKC- dominant negative mutant and examined the response ofthe cells to NGF and EGF. To examine the level of protein expression,we analyzed the pooled cultures and three different PKC-overexpressingclones by Western blotting as compared with the vector controls.Fig. 7A
illustrates a representative Western blot of PC12 cellsoverexpressing a PKC- dominant negative mutant compared withthe vector control. The levels of the overexpressed PKC- dominantnegative mutant in the transfected cells were 10-fold higherthan the endogenous PKC-. The morphology of cells overexpressingthe PKC- dominant negative mutant was similar to that of thevector control cells. However, these cells displayed a higherrate of cell growth as compared to the vector control cellsand the parental cell line (53 ± 11.4% increase overcontrol; P < 0.002; n = 5). The addition of NGF induced asmaller increase in neurite outgrowth in the PKC- dominant negativemutant-overexpressing cells as compared to the vector controlcells. This was reflected both in the number of cells expressingneurites (Fig. 7B)
and in the length of the neurites (datanot shown). Cells expressing the PKC- dominant negative mutantalso showed a more transient pattern of MAPK phosphorylationin response to NGF as compared to the control cells (Fig. 7C)
.Thus, the maximal level of phosphorylation persisted only upto 30 min and decreased thereafter. EGF did not induce neuriteoutgrowth in these cells, and its effect on cell proliferationwas not significantly affected.

Fig. 7. Expression of PKC- dominant negative mutant inhibits NGF-induced neurite outgrowth and MAPK activation. Stable transfectants of PC12 cells overexpressing PKC- dominant negative mutant or the control vector (M) were harvested and subjected to SDS-PAGE and Western blot analysis. The membranes were probed with anti-PKC- (A). PC12 cells overexpressing a PKC- dominant negative mutant were treated with NGF for 4 days, and cells were counted and scored for the presence of neurites (B). Cells were stimulated with NGF for different periods of time, and ERK1 and ERK2 phosphorylation was determined as described in the Fig. 5 legend (C). The results are of one representative experiment of five similar experiments. Similar results were obtained with three other clones overexpressing the PKC- dominant negative mutant.

EGFR Expression.
One possible explanation for the ability of EGF to induce neuriteoutgrowth and sustained phosphorylation of MAPK could be anincreased expression of EGFR in the PKC--overexpressing cells.To examine this possibility, we immunoprecipitated EGFR fromPC12 cells overexpressing the different PKC isoforms and fromthe vector control cells. No significant difference in the levelof EGFR was found (Fig. 8)
. Similar results were obtained inthree independent clones and two pooled cultures for each ofthe PKC isoforms examined (data not shown).

Fig. 8. Expression of EGFR in PC12 cells overexpressing PKC-, -, or -. Stable transfectants of PC12 cells overexpressing the different PKC isoforms or the control vector (M) were harvested, and immunoprecipitation of the EGFR was performed as described in "Materials and Methods." The results are of one representative experiment of three similar experiments. Similar results were obtained with three other overexpresser clones.

PKC activation is known to play a role in the differentiationand proliferation of various cell types (19, 20)
. PKC isoformsdiffer with regard to tissue distribution, regulation, and enzymaticproperties, and the vigorous efforts of various groups are beginningto clarify the different biological roles of individual isoforms.Recent studies have suggested that specific PKC isoforms maybe tightly linked with cellular proliferation and differentiationin various systems. For example, PKC- and - have been shownto exert opposite effects on cell proliferation in NIH3T3 cells(24)
. PKC- plays a role in PMA-induced myeloid differentiationin mouse 32D cells (25)
, and PKC- has been linked with neuronaldifferentiation of the neuroblastoma cell line SH-SY5Y (26)
.In this study, we compared the effects of NGF and EGF on specificPKC isoform translocation in PC12 cells and showed that EGFinduces neurite outgrowth in PC12 cells overexpressing PKC-.

Conflicting evidence has been reported regarding the role ofPKC in the induction of neurite outgrowth in PC12 cells. Onone hand, it was suggested that PKC does not play a criticalrole in NGF-induced differentiation of PC12 cells because depletionof PKC by chronic treatment with PMA did not inhibit neuriteoutgrowth (27, 28)
. On the other hand, chronic treatment withbryostatin 1, a specific partial antagonist of PKC, did inhibitneurite outgrowth by NGF (29)
. Similarly, treatment of PC12cells with the PKC inhibitor sphingosine (13)
and the injectionof anti-PKC antibody into PC12 cells (30)
have also been shownto inhibit neurite outgrowth in response to NGF. Consistentwith these studies, different PKC isoforms have been implicatedin the differentiation and proliferation of PC12 cells basedon changes in their expression and cellular distribution afterNGF-induced differentiation or serum-induced proliferation (31,32, 33)
.

We found that the PC12 cells used in our study express PKC-,-ß, -, -, -, -µ, and -. Different expression ofPKC isoforms has been reported in various PC12 clones. Thus,some clones have been shown to express PKC-, -ß, -, -,and -(34)
, whereas others have been reported to express, inaddition, low levels of PKC- mRNA (32)
or PKC-(33)
. Differencesin the pattern or relative expression of PKC isoforms in variousclones may account for the different results obtained regardingthe role of PKC in the differentiation of PC12 cells. Our findingsthat NGF and EGF induced differential translocation of PKC-,-, and - suggested that one of these isoforms may be associatedwith the differential cellular effects of these two factors.Using two different antisense oligonucleotides for PKC-, wefound only a small inhibitory effect on neurite outgrowth byNGF, although the expression of PKC- was significantly reduced.3These results are in contrast with those reported by Colemanand Wooten (35)
and suggested that PKC- or PKC- might be involvedin the differential effect of NGF and EGF on neurite outgrowth.

We found that EGF induced neurite outgrowth in cells overexpressingPKC-, whereas it did not have a significant effect on cellsoverexpressing either PKC- or -. PKC- is expressed in high levelsin the nervous system and has been implicated in neural differentiation(36)
. In the developing chick brain, PKC- is highly expressedin differentiated neurons, and in immunoreactive neurons ithas been localized in axons and presynaptic nerve terminals(37)
. Further support for the role of PKC- in neural differentiationcomes from in vitro studies in which PKC- has been shown tobe involved in neurite outgrowth in human neuroblastoma cells(26)
and in bradykinin-induced neurite outgrowth in PC12 cells.In addition, recent studies reported that PKC- mediates theenhancement of NGF induction of neurites by PMA and ethanolusing cells overexpressing either PKC- or an inhibitory fragmentfrom PKC-(38, 39)
.

In addition to inducing neurite outgrowth in cells overexpressingPKC-, EGF also induced a sustained phosphorylation of MAPK inthese cells. Although NGF and EGF exert different effects onPC12 cells, there are both common and distinct aspects of theirsignaling pathways (6)
. Various studies have demonstrated thatboth NGF and EGF activate p21ras, which is linked to the activatedreceptors by the adaptor proteins Shc and Grb2 (40)
and eventuallyleads to the activation of a kinase cascade including Raf, MAPKkinase, and MAPK (41)
. However, the patterns of phosphorylationinduced by NGF and EGF are quite distinct. Thus, EGF induceda more transient tyrosine phosphorylation of PLC- as comparedto NGF (9)
. Similarly, p21ras, MAPK kinase, MAPK, and phosphatidylinositol3'-kinase are also more rapidly and transiently phosphorylatedand activated in response to EGF as compared to NGF in PC12cells (7, 9, 22)
. Recently, it was suggested that Rap1 mediatesthe differentiation of PC12 cells in response to NGF by sustainedactivation of MAPK via activation of B-Raf. It is currentlynot clear whether PKC- converges on the Rap1-B-Raf pathway orwhether it represents an alternative pathway. Our results furthersupport the hypothesis that the duration of growth factor-inducedactivation of signaling pathways is related to the cellularresponse.

The results reported here suggest that PKC- may be associatedwith activation of the MAPK pathway in PC12 cells. DifferentPKC isoforms have been implicated in the regulation of the MAPKpathway by influencing different upstream and downstream kinases.Because the effect of EGF and NGF on the translocation of PKC-differs in both the kinetics of the effect and the cellulardistribution of the isoform after stimulation, it is possiblethat either the duration of PKC- activation or its localizationin the cell may be of importance in the activation of the MAPKpathway. Indeed, PKC- has been reported to phosphorylate Rafand to induce a persistent activation of MAPK in fibroblasts(42)
. Thus, it is possible that EGF activation of PKC- in theoverexpressers activates one of the upstream kinases that leadsto increased MAPK activation. In addition, NGF has been reportedto activate PKC- in PC12 cells (43)
and to induce the bindingof PKC- to F-actin in nerve terminals (44)
, thus suggestinga possible role for this interaction in neurite outgrowth. Oneof the most striking differences in the effects of NGF and EGFon PKC- was the ability of NGF to induce a massive translocationof this isoform to the cytoskeleton, whereas EGF induced a transienttranslocation to the membrane. Because we found that there wasan increased expression of PKC- in the cytoskeletal fraction(data not shown) in cells overexpressing PKC-, it is possiblethat the presence of this isoform in the cytoskeleton may playa role in the differentiative effect of EGF.

Although EGF has been reported to act as a mitogen in PC12 cells,it has also been shown to induce cell differentiation undercertain conditions. These conditions are associated with treatmentsthat increase the expression of the EGFR or induce a sustainedtyrosine phosphorylation and a decreased down-regulation ofthe receptor (45, 46)
. In addition, cell growth arrest bycAMP (47)
, activation of the signal transducers and activatorsof transcription tyrosine kinase pathway (48)
, and changesin the pattern of MAPK activation (49)
have been also suggestedto play a role in the induction of neurite outgrowth by EGF.Our results indicate that overexpression of PKC- is anothermechanism that converts the mitogenic effect of EGF to a differentiativeone. EGF-induced differentiation of these cells may be relatedto the observed reduction in cell proliferation, which is similarto the effect obtained in cAMP-treated cells (47)
, or to thesustained activation of MAPK via PKC-. Interestingly, cAMP wasrecently shown to induce the translocation of PKC- in PC12 cells,thus providing further support for the importance of this isoformin PC12 cell differentiation (50)
.

The present results also suggest a role for PKC- in neuriteoutgrowth in response to NGF, because expression of a PKC- dominantnegative mutant reduced the ability of NGF to induce both neuriteoutgrowth and MAPK phosphorylation. Our results differ fromthose reported by Hundle et al.(38)
, who concluded that inhibitionof PKC- does not affect the ability of NGF to induce neuriteoutgrowth but rather abrogates the enhancement of the NGF effectexerted by ethanol and PMA. The differences in our results mayarise from the use of different PC12 clones that express variouslevels of specific PKC isoforms or from different approachesfor studying the role of PKC.

In conclusion, the results of this study suggest that PKC- providesa positive signal for the neurite outgrowth response by NGFand EGF via activation of the MAPK pathway. Our findings providean important system for analyzing the signals involved in neuriteoutgrowth and the interactions of PKC with the MAPK pathway.

The altered sites introduced into primers A and B (underlinednucleotides) were designed to mutate the original amino acidsLys437 and Val438 to Arg437 and Ala438, respectively, and tointroduce a new ApaI restriction cutting site (GGGCCC). Themutant cDNA fragment generated by the PCR overlap extensionprocedure was cloned into the mammalian epitope-tagging expressionvector, which contains the metallothionein promoter (53)
. ThePCR reactions were performed with low (10 or less) cycle numbersusing the high-fidelity Vent DNA polymerase to minimize thechance of undesired point mutations. The introduced ATP bindingsite mutation was verified in selected clones by restrictiondigestions and DNA sequencing.

PC12 Cultures and Cell Transfection.
PC12 cells originally obtained from Dr. G. Guroff (NIH, Bethesda,MD) were grown in medium consisting of DMEM containing 10% heat-inactivatedhorse serum, 5% FCS, 2 mM glutamine, 50 units/ml penicillin,and 0.05 mg/ml streptomycin in a 10% CO2 atmosphere. The cellswere transfected with either the empty vector or with the differentPKC expression vectors using LipofectAMINE (Life Technologies,Inc.) according to the procedure recommended by the manufacturer.The transfected cells were grown in selection medium containing450 µg/ml G418 (Life Technologies, Inc.). After 23weeks in selection medium, single colonies were picked, expanded,and screened for the presence of different PKC isoforms usingWestern blot analysis. Experiments were routinely carried outon pools of transfected cells, but all of the results were confirmedon three different individual clones for each of the differentisoforms.

Neurite Outgrowth Assay.
For studies of neurite outgrowth, cells were plated in collagen-coated6-well plates at a density of 1 x 104 cells/ml in either growthmedium or DMEM containing 2% horse serum. After 24 h, cellswere treated with the appropriate factor, and the appearanceof neurites was examined at different times. Cells bearing neuritesat least twice the size of the cell body were counted.

Immunoprecipitation.
PC12 cells overexpressing the different PKC isoforms or thecontrol vector were washed three times with cold PBS and scrapedinto 1 ml of lysis buffer containing 50 mM HEPES (pH 7.5), 150mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 10 µg/mlaprotinin, 10 µg/ml leupeptin, 1 µM 4-(2-aminoethyl)benzenesulfonylfluoride, and 1 mM EDTA. After mixing, the samples were incubatedon ice for 30 min and then centrifuged in a microcentrifugeat 4°C for 5 min. The supernatant was removed and preabsorbedwith 25 µl of protein A/G-Sepharose (50%) for 10 min.The samples were then spun at 4°C for 3 min at 15,000 xg, and the supernatants were taken for immunoprecipitation.Immunoprecipitation was performed by rotating the samples overnightwith 4 µg/ml anti-EGFR antibody and 30 µl of proteinA/G-Sepharose at 4°C. The samples were spun at 15,000 xg at 4°C for 3 min and washed three times with radioimmunoprecipitationassay buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1%Triton X-100, 0.1% SDS, and 1% deoxycholate. The pellets wereresuspended in 25 µl of SDS sample buffer and boiled for5 min. Before SDS-PAGE, samples were centrifuged again as describedabove, and all of the supernatants were subjected to Westernblotting.

Immunoblot Analysis.
Lysates (20 µg of protein) were resolved by SDS-PAGE (10%)and transferred to nitrocellulose membranes. To determine theprotein content of individual lanes, membranes were stainedwith 0.1% Ponceau S solution in 5% acetic acid. The PonceauS staining was removed by several washes with PBS (pH 7.4);the membranes were blocked with 5% dry milk in PBS and subsequentlystained with the primary antibody. Specific reactive bands weredetected using a goat antirabbit or goat antimouse IgG conjugatedto horseradish peroxidase (Bio-Rad, Hercules, CA), and the immunoreactivebands were visualized by the enhanced chemiluminescence Westernblotting detection kit (Amersham, Arlington Heights, IL). Thespecificities of the antibodies (for all PKC isoforms) weredetermined by the use of soluble immunogenic peptides that competewith the epitope of the correct molecular size of the specificPKC isotypes immobilized on the membranes.

MAPK Activation.
Activation of MAPK was analyzed using a rabbit polyclonal phospho-specificMr 42,000/44,000 MAPK antibody directed against the phosphorylatedform of the MAPK enzyme (Thr183 and Tyr185). For this assay,cells were serum-starved for 1 h and then stimulated with eitherNGF or EGF for different periods of time. Cells were washedin ice-cold PBS and harvested in lysis buffer, and cell lysateswere subjected to Western blot analysis. Membranes were stainedwith anti-ACTIVE MAPK antibody followed by antirabbit antibodyconjugated to horseradish peroxidase. The immunoreactive bandswere visualized by the enhanced chemiluminescence Western blottingdetection kit (Amersham).

[3H]PDBu Binding.
[3H]PDBu binding was measured using the polyethylene glycolprecipitation assay (54)
. Briefly, cell lysates (460µg of protein/assay) were incubated with 20 nM [3H]PDBuin the presence of phosphatidylcholine/phosphatidylserine (80:20).Nonspecific binding, which was determined in the presence of30 µM nonradioactive PDBu, was subtracted to give specificbinding. Data represent triplicate determinations in each experiment.

Cell Proliferation Assay.
Cells overexpressing the different PKC isoforms or the controlvector cells were seeded in two sets of triplicate wells in96-well plates and incubated in the absence or presence of NGFor EGF for a period of 4 or 6 days. Cells were labeled withBrdUrd for the last 6 h in culture. Cells were washed threetimes and fixed, and the DNA was denatured. The cells were thenincubated with anti-BrdUrd antibody conjugated with horseradishperoxidase for 2 h. The immune complexes were detected usingthe substrate tetramethylbenzidine, and the reaction productwas quantified by measuring the absorbance at 450 nm with areference wavelength of 690 nm.

Footnotes

The costs of publication of this article were defrayed in partby the payment of page charges. This article must thereforebe hereby marked advertisement in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

Reinhold D. S., Neet K. E. The lack of a role for protein kinase C in neurite extension and in the induction of ornithine decarboxylase by nerve growth factor in PC12 cells. J. Biol. Chem., 264: 3538-3544, 1989.[Abstract/Free Full Text]